Disruption of gene mg218 of Mycoplasma genitalium through homologous recombination leads to an adherence-deficient phenotype

Abstract

Although the complete genome of Mycoplasma genitalium has been sequenced, the functional identification of various genes, including those involved in virulence, has not been accomplished. Further compounding these difficulties has been the failure to develop genetic tools in mycoplasmas that permit the assessment of gene and operon function and regulation. To determine whether homologous recombination could be developed as a tool to analyze the function of genes in M. genitalium, a plasmid that replicates in Escherichia coli but not in M. genitalium was constructed to disrupt the cytadherence-related gene mg218 of M. genitalium. The electroporation of this disruption plasmid into wild-type hemadsorption-positive (HA+) M. genitalium cells permitted the isolation of HA- (strain JB1) and partial HA+ (strains JB2 and JB20) transformants. Analysis of the transformants by Southern hybridization indicated that homologous recombination occurred at the mg218 locus by single-crossover events in JB1 and JB2 and by a double-crossover event in JB20. While integration of the disruption construct abolished the expression MG218 in JB1, strains JB2 and JB20 exhibited a truncated MG218 protein (160 kDa), possibly because of in-frame fusion of the disrupted mg218 gene with sequences downstream of the gentamycin-resistance gene present in the disruption construct. Strain JB1, which lacked MG218, displayed a post-translational defect, being unable to maintain the structural integrity of the major adhesin P140 and its operon-related protein P110, in contrast to JB2 and JB20. It appears that MG218 influences the stability of other cytadherence-related proteins in vivo. Thus, targeted gene disruption through homologous recombination will be a powerful and promising tool for investigating the biology and pathogenesis of M. genitalium.

Figure 1

Schematic representation of the derivation of the mg218 disruption construct. (A) The mg218 locus in the M. genitalium chromosome. Restriction sites are based on the DNA sequences available in National Center for Biotechnology Information databases with accession no. U39699. The hatched box represents the coding region of mg218 and the stippled boxes represent the flanking regions of mg218. The small black boxes represent the location of the primers used for amplifying a portion of mg218 DNA. The arrow indicates the direction of transcription of mg218. (B) Schematic representation of plasmid pDP218. The hatched box represents the 2-kb PCR fragment encompassing the 5′ portion of mg218. The open box represents the pCR2 vector. (C) Schematic representation of plasmid pDP219. The hatched boxes indicate the mg218 DNA. The densely stippled box indicates the gentamycin-resistance gene from Tn4001 inserted at the StuI site of pDP218. The open box represents the pCR2 vector. A detailed description of the construction of plasmids pDP218 and pDP219 appears in Materials and Methods. EI, EcoRI; EV, EcoRV; H, HindIII; K, KpnI; P, PstI; SI, SalI; Sp, SpeI; St, StyI; Su, StuI.

Figure 2

SDS/PAGE and Western blot profiles of M. genitalium wild-type G37 and mg218 mutants JB1 and JB2 (strains JB2 and JB20 showed similar protein profiles; hence, only JB2 is shown). (A) Total proteins separated by SDS/7.5% PAGE and stained with Coomassie brilliant blue. (B) Western blot probed with anti-MG218 antibodies identifying intact and truncated MG218. (C) Western blot reprobed with anti-P140 and anti-P110 antibodies. MG218, P140, and P110 represent the corresponding M. genitalium proteins. Marker proteins are indicated on the extreme left and right.

Figure 3

Southern hybridization profiles of DNA from M. genitalium strains. G37, wild-type; JB1 and JB2, mg218 mutants. StyI, SpeI, and SpeI+StyI represent the restriction enzymes used to cut genomic DNA. DNA fragments were resolved in 0.8% agarose gel, Southern transferred to nitrocellulose membrane, and probed with the following: a 2-kb mg218 gene fragment (A; probe I); a 2.5-kb gentamycin gene fragment (B; probe II); and a 3.9-kb pCRII plasmid DNA (C; probe III). The sizes of the DNA bands are indicated in kilobases (kb).

Figure 4

Southern hybridization profiles of DNA from M. genitalium strains. G37, wild-type; JB20, mg218 mutant. StyI, SpeI, and SpeI+StyI represent the restriction enzymes used to cut genomic DNA. DNA fragments were resolved in 0.8% agarose gel, Southern transferred to nitrocellulose membrane, and probed with the following: a 2-kb mg218 gene fragment (A; probe I); or a 2.5-kb gentamycin gene fragment (B; probe II). There was no positive signal with 3.9-kb pCR2 plasmid DNA (probe III) with JB20 DNA. Arrows indicate the hybridization signals due to similar size fragments of 3.2 kb and 3.3 kb. The sizes of the DNA bands are indicated in kilobases (kb).

Figure 5

Homologous recombination at the mg218 locus in M. genitalium. (A) Schematic representation of the two possible crossover events (X₁ and X₂) of pDP19 (mg218 disruption construct) with the mg218 locus in the genomic DNA of M. genitalium. (B) mg218 locus and its flanking regions in the wild-type M. genitalium genome. (C) mg218 locus disrupted by single crossover event X₁ in the M. genitalium mutant strain JB1. (D) mg218 locus disrupted by single crossover event X₂ in the M. genitalium mutant strain JB2. (E) mg218 locus disrupted by double crossover events X₁ and X₂ in the M. genitalium mutant strain JB20. Hatched boxes, mg218 DNA region; lightly stippled boxes, flanking regions of mg218 locus; densely stippled box, gentamycin-resistance gene; open box, pCR2 plasmid DNA. The different sizes of SpeI and StyI fragments observed in Southern blotting are represented below the mg218 locus of each strain. EI, EcoRI; EV, EcoRV; H, HindIII; K, KpnI; P, PstI; Sp, SpeI; St, StyI; Su, StuI.

Figure 6

Pulse–chase analysis of M. genitalium proteins. (A) Total protein profiles of wild-type strain G37 at 0 h, 1 h, 2 h, and 5 h. (B) Total protein profiles of mg218 mutant strain JB1 at 0 h, 1 h, 2 h, and 5 h. MG218, P140, and P110 represent the corresponding M. genitalium proteins. The pulse with [³⁵S]methionine/[³⁵S]cysteine was for 3 h, and individual samples were removed at 1-h intervals for up to 5 h. Then, mycoplasma proteins were separated by SDS/5% PAGE, and the gels were dried prior to autoradiography.